In order to find a specific nucleic acid or gene, a method is commonly employed that amplifies a target nucleic acid or gene and detects the product of amplification. As the methods that specifically amplify a target nucleic acid or gene, the following methods are known. PCR method (see Non-Patent Documents 1 and 2, for example), RT-PCR method (see Non-Patent Documents 1 and 2, for example), ICAN method (see Patent Document 1, for example), LAMP method (see Non-Patent Document 3, for example), RCA method (see Non-Patent Document 4, for example), and primer extension method (see Non-Patent Document 5, for example). Among these examples, PCR and RT-PCR are most commonly used. In these methods, short nucleic acid sequences including the base sequence of the target nucleic acid are used as primers, and a template-specific nucleic acid synthesizing reaction is performed in vitro, using DNA polymerase or RNA polymerase.
If the amplified nucleic acid fragments were labeled by suitable methods during or after the amplification reaction, these nucleic acid amplification methods are able to detect even a trace amount of nucleic acid in a sample. Other known examples include DNA microarray (macroarray) method and differential display method. For the detection of nucleic acid, these methods use primers of random base sequences to non-specifically amplify and label the nucleic acids. Recently, DNA microarray has caught a particular attention for its ability to comprehensively detect genes that are associated with various types of diseases.
In addition to these nucleic acid amplification methods, ISH (in situ hybridization) method and FISH (fluorescein in situ hybridization) method are available. In order to find a specific nucleic acid or gene, these methods cause a target nucleic acid in tissues or cells to hybridize with probes, for which labeled nucleic acids having the complementary base sequences are used (see Non-Patent Document 1, for example). ISH method is widely used to detect expression of specific genes in tissues, or compare expression levels of specific genes in tissues. FISH method is widely used to find specific gene regions on the chromosomes.
Another example is in situ PCR method. This method employs PCR to amplify the target nucleic acid, uses ISH to cause hybridization with probes, and detects the target nucleic acid with a microscope (see Non-Patent Document 2, for example). However, owning to difficulties in setting optimum reaction conditions, the method suffers from poor reproducibility and is not pervasive.
Applicant of the present invention has been marketing “HYBRISEP®”, which is a bacteria detecting kit for detecting bacteria in peripheral blood white blood cells according to the ISH method (in vitro diagnostic drug (Approval Number: AMZ00620000)). “HYBRISEP®” has come under the spotlight in the field of infection, because it is able to detect bacteria at about 4 times more sensitivity than conventional blood culture methods and thereby complete the test within a day, instead of at least 3 days conventionally required (see Non-Patent Document 6, for example). Further, Applicant has proposed a method for detecting and identifying foreign microbes phagocytosed by phagocytes (see Patent Document 2, for example), and improvement of the method (see Patent Document 3, for example). “HYBRISEP®” was developed based on these inventions.
[Patent Document 1]    Japanese Patent No. 3433929 (registered on May 30, 2003, issued on Aug. 4, 2003)
[Patent Document 2]    International Publication WO89/10411 (published on Nov. 2, 1989, corresponding patent: Examined Patent Publication No. 07-40)
[Patent Document 3]    International Publication WO02/099133 (published on Dec. 12, 2002)
[Non-Patent Document 1]    J. Sambrook et al. “Molecular Cloning, A Laboratory Manual, Third Edition” Cold Spring Harbor Laboratory (2001)
[Non-Patent Document 2]    Hisaji Maki, “PCR Tips-Techniques and Hints for Mastering PCR-, Shujunsha, 1999
[Non-Patent Document 3]    Tsugunori Notomi et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, vol. 28, No. 12: e63 (2000)
[Non-Patent Document 4]    Lizardi P M et al. Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nature Genetics, July; 19(3):225-32. (1998)
[Non-Patent Document 5]    B. D. Hames, S. J. Higgins: Translation by Masami Horikoshi, “Gene Expression and Transcription Factors”, Medical Science International, 1996
[Non-Patent Document 6]    Akio Matsuhisa, Hiromasa Araki, “Clinical Use of In Situ Hybridization in Sepsis Diagnosis”, BIO Clinica, Hokuryukan, 1999, Vol. 14, No. 1, p. 97-101
While the foregoing nucleic acid amplifying methods such as PCR and hybridization methods such as ISH are used to detect specific nucleic acids or genes, the methods may not be able to provide enough detection sensitivity, reproducibility, or convenience, etc., when the sample contains only a trace amount of target nucleic acid or gene.
In the nucleic acid amplifying method such as PCR, nucleic acids need to be extracted from the sample. In this process, it is difficult to extract the nucleic acids without any loss. For example, there are cases where the amount of nucleic acid collected from the sample may not be sufficient to provide a template due to a loss in the extracting procedure, even when the sample contains a sufficient amount of target nucleic acid to be used as a template for amplification. In such case, the target nucleic acid may not be amplified sufficiently due to reduced amplification efficiency, with the result that the target nucleic acid in the sample cannot be detected. In this case, the result is false negative and inaccurate. Further, even the same sample may yield different results depending on how much nucleic acid is lost in the extracting procedure. This works against reproducibility. The problem of nucleic acid amplifying method that requires extraction of nucleic acids, then, is that a loss of nucleic acid in the nucleic acid extracting procedure may lead to reduction of amplification efficiency and detection sensitivity when the sample contains only a trace amount of target nucleic acid.
Further, when the reagent contains substances that inhibit amplification reaction (for example, heparin, detergent, protein denaturing agent, organic solvent, etc.), the problem of reduced amplification efficiency and detection sensitivity may also occur.
As for the hybridization method such as ISH, the method does not require extraction of nucleic acid and there accordingly will be no loss of nucleic acid. However, when the sample contains only a trace amount of target nucleic acid, there is a difficulty in detecting probe nucleic acids that have hybridized with the target nucleic acid.
While “HYBRISEP®” developed by the Applicant of the present invention enables accurate and quick detection of bacteria in peripheral blood white blood cells according to the ISH method, it has the following drawbacks.
(1) Certain skill is required because signaling of the cells is observed by naked eyes through a microscope.
(2) Detection rate is reduced in clinical samples obtained from patients with a reduced number of white blood cells, because the “HYBRISEP®” detects only those bacteria phagocytosed by the white blood cells.
The present invention was made in view of the foregoing problems, and an object of the present invention is to provide a nucleic acid detection method that can accurately and quickly detect a target nucleic acid even when a sample contains only a trace amount of target nucleic acid.